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Abstract

It has recently been reported that tomato fruit chromoplasts can synthesize ATP de novo using an ATP synthase complex harboring an atypical γ-subunit which is also present in a variety of plant species. However many aspects related with the biochemical processes underlying this process remain largely unknown. Here we describe detailed protocols for the isolation of tomato fruit chromoplasts and the determination of ATP levels (end-point measurements) and ATP synthesis rates (kinetic measurements) in these organelles using bioluminescent luciferin/luciferase based assays.

Aliquot the resuspended chromoplasts into microcentrifuge tubes (30 µl per tube) and incubate samples at room temperature (23 °C) on a bench orbital shaker for as long as needed according to the experiment (usually 10 to 60 min). This step allows chromoplasts to perform the biochemical reactions needed for the de novo synthesis of ATP. At the end of the incubation, samples can be processed immediately or stored at -80 °C.

Add 90 µl of the “Cell Lysis Reagent” (contained in the kit “ATP Bioluminescence Assay Kit HS II”) to each tube, mix and incubate the samples for 2 min at 95 °C in a heat block, then transfer the samples on ice.

Centrifuge the samples for 3 min in a microcentrifuge at high speed and room temperature to pellet chromoplasts debris. Transfer the supernatants to fresh microcentrifuge tubes. Keep samples on ice.

Transfer 50 µl from each sample to a 96-well microplate. Keep samples on ice.

Perform the ATP determination reactions using the Glomax 96 Microplate Luminometer. For these experiments only one injector is used, primed with the “Luciferase reagent” included in the kit “ATP Bioluminescence Assay Kit HS II” and prepared in advance following the instructions provided by the supplier. The instrument is set to add automatically 50 µl of the “Luciferase reagent” to each sample and to start the measurements after 1 sec delay. The final outcome values of the instrument show relative luminescence (light) of the samples, which expresses ATP content according to the reaction: D-luciferin + ATP + O2 → oxyluciferin + PPi + AMP + CO2 + light. The values of the blank samples (samples without chromoplasts, but only the buffer C) have to be subtracted from the sample values.Note: The above described method using the “ATP Bioluminescence Assay Kit HS II” kit allows the determination of relative ATP values and the comparison of the ATP content of different samples according to the luminescence observed values. Furthermore, absolute ATP values can be measured using the same kit and method. For this purpose, the ATP standard (contained in the kit) has to be prepared, according to the kit manual, in serial dilutions of known ATP concentration. Next, the luminescence of these serial dilutions has to be determined using the Glomax 96 Microplate Luminometer to generate an ATP standard curve. Comparison of the luminescence values of the samples with those of the ATP standard curve can help for the estimation of absolute ATP values of the samples of interest. The estimated ATP amount can be referred to chromoplast protein content (see step D).

Measurement of ATP synthesis rates (kinetics measurements)

Reconstitute the “ENLITEN® rLuciferase/Luciferin Reagent” following the instructions provided by the supplier. The reagent has to be stored at -20 °C in 2 ml aliquots for no more than two weeks.

Resuspend chromoplasts (step A21) in 500 µl of buffer D by gentle pipetting. Keep chromoplast samples on ice and start the ATP measurements within the first 15 min after chromoplast isolation.

Allow the following reagents to equilibrate at room temperature for approximately 5 min. Then, add the reagents in this order into the wells of a 96-well microplate:

rLuciferase/Luciferin Reagent

80 µl

Buffer E

80 µl

DAPP

4 µl

NADH 100 mM and NAD+ 100 mM
(or NADPH 100 mM and NADP+ 100 mM)

2 µl of each

Chromoplasts resuspended in buffer D

20 µl

Note: DAPP is an inhibitor of adenylate kinase that does not affect the ATP synthase.

Place the microplate in the luminometer and start the “kinetic protocol”. The injector should inject automatically 40 µl of 0.5 mM ADP and light emission data should be collected during 60 sec for each well.

To prepare the ATP calibration curve, select the “non-injection kinetic protocol” in the luminometer and adjust the following settings: i) delay in the first measurement: 0 sec, and ii) integration time: 30 sec.

Add the following components in the appropriate wells of the microplate (at room temperature):

rLuciferase/luciferin Reagent

80 µl

Buffer

80 µl

Ultrapure water

20 µl

DAPP

4 µl

Corresponding ATP standard (10 µM, 0.1 µM or 0.001 µM ATP)

20 µl

Chromoplasts resuspended in buffer D

20 µl

Note: Chromoplasts are added because they quench part of the light emitted in the luciferase reaction.

Place the plate in the luminometer and start the “non-injection kinetic protocol”.

Data analysis: Relative luminescence data will appear in an Excel spreadsheet. Calculate the ATP standard curve equation using the Excel functions and apply this equation to obtain the ATP concentration of all samples at each measurement time (sec). Then, the ATP synthesis rate can be represented in a graph (Y-axis: nmol ATP; X-axis: sec) and can be expressed as nmol ATP x mg protein-1 x sec-1 performing the appropriate calculations. Note: Data from the first 5 sec of kinetics measurements are discarded because they are usually erratic.

Determination of protein content of chromoplast samples

Aliquots of 50 µl of chromoplast samples resuspended in buffer C or D are supplemented with Tween-20 at 1% final concentration and incubated at 4 °C in an orbital shaker for 30 min.

Process the samples using the RC DC Protein Assay kit following the indications provided by the supplier.

This protocol has been adapted from methods previously described in Angaman et al. (2012) and Pateraki et al. (2013). This work has been supported by grants from the Spanish Ministerio de Ciencia e Innovación (BIO2009-09523) and Ministerio de Economía y Competitividad (AGL2013-43522-R), both including FEDER Funds, the Spanish Consolider-Ingenio 2010 Program (CSD2007-00036 Centre for Research in Agrigenomics) and the Generalitat de Catalunya (2009SGR0026). Marta Renato is a recipient of a predoctoral fellowship from the Spanish Ministerio de Educación, Cultura y Deporte.

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